Interaction between nuclear localization signal-retinoic acid receptor alpha and Ubiquilin 1.

Abstract

To identify the interaction between nuclear localization signal-retinoic acid receptor alpha (NLS-RARalpha) and Ubiquilin 1(UBQLN1). The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1, which expressed bait-protein NLS-RARalpha and target protein UBQLN1 respectively, were cotransformated into yeast AH109. The interaction of the expression plasmids in the living cells was investigated by yeast two-hybrid assay. HA-tagged fusion protein (pCMV-HA-NLS-RARalpha) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were constructed, identified, and cotransfected respectively into human embryo kidney 293 cells(HEK293). The interaction was detected by co-immunoprecipitation. Blue clones were found on yeast AH109 plate cotransformated with pGBKT7-NLS-RARalpha and pACT2-UBQLN1. Double restriction enzyme digestion showed that pCMV-HA-NLS-RARalpha and pCMV-Myc-JTV1 were successfully constructed. Then HA-NLS-RAR protein was immunoprecipitated by anti-HA polyclonal antibody and the expression of Myc-UBQLN1 was tested by Western blot with anti-Myc monoclonal antibody from immunoprecipitated complex. The interaction between NLS-RARalpha and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.